四川山地乌骨鸡IL-2、15、18、IFN-γ基因克隆测序及分子佐剂效应研究
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摘要
鸡白细胞介素2(IL-2)、IL-15、IL-18及干扰素-γ(IFN-γ)是鸡体内重要的细胞因子，在鸡抗病和免疫应答反应中有重要作用。四川山地乌骨鸡是地方优良品种，具有生长快，适应性广、抗病力强等特点。目前有关四川山地乌骨鸡的IL-2、IL-15、IL-18及IFN-γ及其免疫学特性的研究，国内外均未见报道。克隆四川山地乌骨鸡IL-2、IL-15、IL-18及IFN-γ基因，研制分子免疫佐剂及其对疫苗的佐剂效应，提高疫苗免疫应答，有重要学术价值和应用前景。
1．本试验用RT-PCR方法从conA刺激的四川山地乌骨鸡的外周血淋巴细胞总RNA扩增了鸡白细胞介素2及γ-干扰素基因的cDNA。将分离的cDNA片段克隆到PMD18-T载体，进行序列测定分析。结果表明：鸡IL-2基因全部的开放阅读框432bp，与Genbank数据库中的序列进行同源性比较发现：山地乌骨鸡IL-2基因与其他品种鸡IL-2基因核苷酸同源性为98.4-99.5％。鸡IFN-γ基因全部的开放阅读框495bp，与GeneBank上报道的核苷酸序列同源性为97.4％-100％。四川山地乌骨鸡IL-2和IFN-γ克隆在国内外尚未见报道，本试验所克隆的鸡IL-2及IFN-γ基因，丰富了IL-2及IFN-γ基因的资源库，为进一步研究提供了基础材料。
2．用RT-PCR方法从conA刺激四川山地乌骨鸡的脾淋巴细胞总RNA扩增了鸡白细胞介素15基因的cDNA。将分离cDNA片段的克隆到PMD18-T载体，进行序列测定分析。结果表明：鸡IL-15基因基因全部的开放阅读框564bp，与Genbank上序列比较发现，山地乌骨鸡IL-15与其他序列同源性为98.9-99.8％。目前很少关于鸡IL-15的研究报道，四川山地乌骨鸡IL-15克隆在国内外尚未见报道，本试验为进一步研究鸡IL-15的疫苗佐剂效应奠定的基础。
3．本研究应用RT-PCR方法从conA刺激2h后四川山地乌骨鸡的脾脏组织总RNA扩增了IL-18基因的cDNA，将分离cDNA片段的克隆到PMD18-T载体，进行序列测定分析。结果表明：鸡IL-18基因全部的开放阅读框597bp，与GeneBank上报道的核苷酸序列同源性为96.5-100％。其中与Schneider等报道的序列完全一致。四川山地乌骨鸡IL-18克隆在国内外尚未见报道，本研究为鸡IL-18疫苗佐剂效应的研究提供了物质基础。
4．分别双酶切得到IL-2、IFN-γ、IL-15、IL-18 DNA片段，将其亚克隆入真核
chicken interleukin-2(ChIL-2)、 ChIL-15、 ChIL-18 and Chinterferon-γ (ChlFN-γ) play an important role in chicken disease resistance and immune response. Shandi Wugu chicken(SDWG chicken) is a Local chicken breed it had the character of high growth rate、 abroad adaptability and resistance to disease.There were few report on Shandi Wugu chicken IL-2、 IL~15、 IL-18、 IFN-γ and their immunity. Cloning of SDWG chicken IL-2、 IL-15、 IL-18 、 IFN-γ, development of melocular adjuvant and study on the immunenhancemnet for vaccines have great academic value and application prospects.
1.SDWG chicken IL-2 and IFN- γ was amplified by recerse transcription-polymerase chain reaction(RT-PCR)from peripheral blood lymphocytes total RNA stimulated with ConA. Then cloned into PMD18-T vector and sequenced. The result showed that the open reading frame of IL-2 was 432bp. Sharing98. 4-99. 5% homology with the published. The open reading frame of IFN-γ consisted of 495bp, Sharing97. 4-100% homology with the published IFN-γ gene .There is (few) report about the chIL-2 and ChIFN- γ of SDWG chicken. The gene of chIL-2 and ChIFN-γ in our experiment enriched the banks and provided the material for further study.
2. Shandi Wugu ChIL-15 was amplified by RT-PCR from spleen lymphocytes total RNA stimulated with ConA. Then cloned into PMD18-T vector and sequenced. The result showed that the open reading frame of IL-15 was 564bp,
1．本试验用RT-PCR方法从conA刺激的四川山地乌骨鸡的外周血淋巴细胞总RNA扩增了鸡白细胞介素2及γ-干扰素基因的cDNA。将分离的cDNA片段克隆到PMD18-T载体，进行序列测定分析。结果表明：鸡IL-2基因全部的开放阅读框432bp，与Genbank数据库中的序列进行同源性比较发现：山地乌骨鸡IL-2基因与其他品种鸡IL-2基因核苷酸同源性为98.4-99.5％。鸡IFN-γ基因全部的开放阅读框495bp，与GeneBank上报道的核苷酸序列同源性为97.4％-100％。四川山地乌骨鸡IL-2和IFN-γ克隆在国内外尚未见报道，本试验所克隆的鸡IL-2及IFN-γ基因，丰富了IL-2及IFN-γ基因的资源库，为进一步研究提供了基础材料。
2．用RT-PCR方法从conA刺激四川山地乌骨鸡的脾淋巴细胞总RNA扩增了鸡白细胞介素15基因的cDNA。将分离cDNA片段的克隆到PMD18-T载体，进行序列测定分析。结果表明：鸡IL-15基因基因全部的开放阅读框564bp，与Genbank上序列比较发现，山地乌骨鸡IL-15与其他序列同源性为98.9-99.8％。目前很少关于鸡IL-15的研究报道，四川山地乌骨鸡IL-15克隆在国内外尚未见报道，本试验为进一步研究鸡IL-15的疫苗佐剂效应奠定的基础。
3．本研究应用RT-PCR方法从conA刺激2h后四川山地乌骨鸡的脾脏组织总RNA扩增了IL-18基因的cDNA，将分离cDNA片段的克隆到PMD18-T载体，进行序列测定分析。结果表明：鸡IL-18基因全部的开放阅读框597bp，与GeneBank上报道的核苷酸序列同源性为96.5-100％。其中与Schneider等报道的序列完全一致。四川山地乌骨鸡IL-18克隆在国内外尚未见报道，本研究为鸡IL-18疫苗佐剂效应的研究提供了物质基础。
4．分别双酶切得到IL-2、IFN-γ、IL-15、IL-18 DNA片段，将其亚克隆入真核
chicken interleukin-2(ChIL-2)、 ChIL-15、 ChIL-18 and Chinterferon-γ (ChlFN-γ) play an important role in chicken disease resistance and immune response. Shandi Wugu chicken(SDWG chicken) is a Local chicken breed it had the character of high growth rate、 abroad adaptability and resistance to disease.There were few report on Shandi Wugu chicken IL-2、 IL~15、 IL-18、 IFN-γ and their immunity. Cloning of SDWG chicken IL-2、 IL-15、 IL-18 、 IFN-γ, development of melocular adjuvant and study on the immunenhancemnet for vaccines have great academic value and application prospects.
1.SDWG chicken IL-2 and IFN- γ was amplified by recerse transcription-polymerase chain reaction(RT-PCR)from peripheral blood lymphocytes total RNA stimulated with ConA. Then cloned into PMD18-T vector and sequenced. The result showed that the open reading frame of IL-2 was 432bp. Sharing98. 4-99. 5% homology with the published. The open reading frame of IFN-γ consisted of 495bp, Sharing97. 4-100% homology with the published IFN-γ gene .There is (few) report about the chIL-2 and ChIFN- γ of SDWG chicken. The gene of chIL-2 and ChIFN-γ in our experiment enriched the banks and provided the material for further study.
2. Shandi Wugu ChIL-15 was amplified by RT-PCR from spleen lymphocytes total RNA stimulated with ConA. Then cloned into PMD18-T vector and sequenced. The result showed that the open reading frame of IL-15 was 564bp,
引文
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